The hexose monophosphate (or pentose phosphate) pathway and cytosolic isocitrate dehydrogenase (IDP2) have been found to be essential adjuncts to catalase function in preventing intracellular accumulation of the hydrogen peroxide generated during beta-oxidation of fatty acids in peroxisomes. The absence of these two cytosolic sources of NADPH results in lethality of yeast cells transferred to media with fatty acid carbon sources. These observations, peroxisomal metabolism as a source of lethal levels of hydrogen peroxide and a requirement for cytosolic NADPH to prevent lethality, are novel observations in the area of cellular oxidative stress. Further investigation of these phenomena is proposed because oxidative damage to cellular macromolecules has been clearly associated with degenerative diseases and with the process of aging. Aims of this proposal are: (A) Similar requirements for cytosolic NADPH in removal of oxidative byproducts generated during mitochondrial respiration will be examined using a collection of Saccharomyces cerevisiae mutants with combinations of disruptions in genes encoding these and other enzymes with specific antioxidant functions. (B) The heritability of damage to specific genes and cellular proteins will be assessed using genetic crosses of yeast mutant strains that exhibit similar growth phenotypes as a result of endogenous oxidative damage. (C) The unique antioxidant functions of IDP2 will be explored by replacement with homologous yeast isozymes and by using yeast two-hybrid assays to test and screen for specific interactions with other cellular proteins. (D) Patterns of expression and cellular localization of mammalian IDP2 will be examined to clarify possible roles in support of peroxisomal beta- oxidation and/or in provision of NADPH for antioxidant functions.